Acetylcholinesterase (AChE) hydrolyzes acetylcholine at the neuromuscular junction of vertebrates and, thus, plays an important role in neuromuscular transmission. AChE exists in globular forms, G1, and G2, and G4, which consist of 1, 2, or 4 catalytic subunits, and in asymmetric forms, A4, A8, and A12, which consist of 1, 2, or 3 globular tetramers attached to a collagenous tail. This proposal concerns the characterization and purification of the globular G4 AChE from the skeletal muscle of the lamprey Petromyzon marinus. G4 AChE from various species of vertebrates is found in both membrane-bound and soluble forms. Although the membrane-bound enzyme is exclusively amphipathic, the soluble enzyme appears to exist as either an amphipathic or a hydrophilic protein. It has been suggested that the soluble amphipathic AChE may be converted to hydrophilic AChE by proteolysis. This possibility will be investigated through experiments performed with G4 AChE in crude extracts of muscle and with purified enzyme. The amphipathicity of AChE in crude extracts and its modification by proteolysis will be examined by a combination of differential extraction, velocity sedimentation on sucrose gradients, charge-shift electrophoresis, and controlled proteolytic digestion. G4 AChE from lamprey muscle will be purified by affinity chromatography. The purity and molecular weight of the enzyme will be evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Velocity sedimentation and gel exclusion chromatography in the presence and absence of a non-denaturing detergent will provide information about sedimentation coefficient, Stokes radius, molecular weight, and amphipathic nature of the purified AChE, as well as modification of these properties by proteolytic digestion. It appears that G4 AChE from bovine brain is attached to membrane in a unique fashion, with the catalytic tetramers arranged around a small non-catalytic membrane-binding subunit in a pseudo-asymmetric arrangement. To determine if G4 from lamprey is inserted in the membrane similarly, the purified enzyme will be labeled with 125 I-TIDS, a reagent that labels the hydrophobic membrane-binding domains of amphipathic proteins. The turnover number and substrate specificity of the purified enzyme will also be determined. Because of the position of lamprey on the phylogenetic scale, these kinetic parameters should be of interest with respect to the evolution of AChE. These studies will provide a detailed picture of the structure and function of the G4 AChE from vertebrate skeletal muscle.